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  • Title: Enzyme distribution derived from macroscopic particle behavior of an industrial immobilized penicillin-G acylase.
    Author: van Roon JL, Joerink M, Rijkers MP, Tramper J, Schroën CG, Beeftink HH.
    Journal: Biotechnol Prog; 2003; 19(5):1510-8. PubMed ID: 14524713.
    Abstract:
    The macroscopic kinetic behavior of an industrially employed immobilized penicillin-G acylase, called Assemblase, formed the basis for a discussion on some simple intraparticle biocatalytic model distributions. Assemblase catalyzes the synthesis of the widely used semisynthetic antibiotic cephalexin. Despite the obvious advantages of immobilization, less cephalexin and more of the unwanted byproduct d-(-)-phenylglycine are obtained due to diffusional limitations when the immobilized enzyme is employed. To rationally optimize Assemblase, the parameters particle size, enzyme loading, and enzyme distribution, which severely determine the macroscopic particle performance, were studied on the basis of macroscopic observations. Laser diffraction measurements showed that the particle sizes in Assemblase vary as much as 100-fold. The relative and total enzyme loadings in Assemblase and fractions thereof of different sizes were determined by initial-rate d-(-)-phenylglycine amide hydrolysis, cephalexin synthesis experiments, and active-site titration. These experiments revealed that the loading of penicillin-G acylase in Assemblase was inversely correlated with the particle diameter. Apart from enzyme loadings, estimates on the intraparticle enzyme distribution came from cephalexin synthesis experiments, where mass-transport limitations were present. Although this method cannot provide the level of detail of specific labeling experiments, it is simple, fast, and cheap. Within the set of simple model predictions, a heterogeneous enzyme distribution with most biocatalyst present in the outer region of the particle (within the outer 100 microm) gave the best description of the observed behavior, although no exact correlation was established. Highly detailed determination of intraparticle enzyme distributions must come from immunolabeling.
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